Activities of endopeptidases (EPs) of five specificities and aminopeptidases (APs) of five specificities have been studied in soluble extracts (cytosols) of benign and malignant human gynecologic specimens and a cell line derived from a mixed mesodermal uterine tumor (SK-UT-1). Results were compared with analogous studies of human leukemic cells. Fluorometric assays using derivatives of 7-amino-4-methylcoumarin (AMC) required at most 0.02 ml of cytosol. The size and shape of the enzymes were evaluated by assays following centrifugation and gel filtration. Results include comparisons of relative activities (RAs) of each specimen on various substrates and effect of enzyme activators and inhibitors. Aminopeptidase activities on neutral substrates (leu-, ala-, or phe-AMC) were reduced to less than 20% of control levels by 1 mM puromycin in all systems tested. Cleavage of a basic substrate (arg-AMC) was either slightly reduced or elevated by puromycin but was stimulated 10-fold by physiological salt concentration. Among the EP substrates tested, highest activities were detected with auc-(leu)2-val-tyr-AMC (tyr) and boc-val-leu-lys-AMC (Lys). The RA of cytosols from SK-UT-1 cells on tyr/lys was consistently about 5, butRAs of gynecologic specimens on these substrates varied widely. The size and shape of EPs and APsfrom SK-UT-1 cells were similar to those of the corresponding enzymes from leukemic cells. Specific aims for this year are: (1) further enzymatic and physical-chemical characterization of three APs and three EPs in SK-UT-1 cells; (2)\studies of effects of culture conditions on enzyme levels in cytosol and other subcellular fractions and on peptidase secretion into the medium; (3)\comparisons of the corresponding enzymes in benign uterine specimens, carcinomas, and sarcomas; (4)\attempts to identify which, if any, of these enzymes is responsible for the apparent cleavage of steroid receptors in extracts of gynecologic tissues; and (5)\evaluation of the clinical significance of these results. (D)